Oral cancer invasion mediated by TGF-β1/MMP via EMT of CSC

Methods: Four OSCC cell lines, SSCC15, CC25, HN5 and Tca8113 were treated with 5 ng/mL of TGF-β1 for up to 3 days, whilst conditioned medium (CM) of these cells was collected for co-culture with osteoblasts (hFOB). Assays of cell proliferation, morphology and CSC tumour-sphere formation were determined: proteolytic activities of MMP-2 and MMP-9, and putative markers of CSC, EMT and osteo-molecules were detected by gelatine zymography, immunohistochemistry, western blotting and real-time PCR respectively. Targeted molecules were examined by in tissue sections of bone-invasive OSCCs.

Results: TGF-β1 had no effect on growth of OSCC cell lines, but mediated the initiation of CSCs, as determined by immunostaining for CD44. Following treatment withTGF-β1, staining of vimentin and EMT markers (Twist-1 and N-cadherin) was found in cancer cells. Zymogenic activities of MMP-2 and MMP-9 were increased in OSCC cells following culture with CM of hFOB. The ratio of receptor activator of nuclear factor ligand (RANKL)/ osteoprotegerin (OPG), zymogen and MMP-9 were increased in hFOB cells cultured with CM from OSCC lines, while zymogen expression of MMP-2 was decreased. In clinical samples, all targeted molecules were expressed in invading malignant keratinocytes, and OPG was expressed in osteoclasts. Osteoclast-related molecules [membrane type 1-MMP (MT1-MMP) and RANKL] were up-regulated, whilst OPG was down-regulated in cancer cells.

Conclusions: This study suggests that EMT of CSC in OSCC is triggered by TGF-β1 and promotes bone invasion of OSCC.